1. Field of the Invention
The present invention relates to a reversed-phase liquid chromatography using a mobile phase containing water as its main component; and a liquid chromatograph apparatus and a column each for use in a reversed-phase liquid chromatography.
2. Related Art Statement
There is known a reversed-phase liquid chromatography for separating a water-soluble compound, using a mobile phase as a solvent that solves a sample to be separated, and a stationary phase as a liquid or a solid that is carried by a packing material (i.e., a carrier) that packs a column. The mobile phase contains water as its main component, and the stationary phase contains a compound having a carbon chain. Here, the mobile phase containing water as its main component is defined as encompassing a 100% water; a buffer that is prepared by adding an agent to water (hereinafter, in the present specification, the term “100% water” is defined as encompassing the buffer, unless otherwise specified); a solution that is prepared by adding, to the 100% water, a salt, an acid, etc. (e.g., sodium phosphate, trifluoroacetic acid, triethylamine, or sodium perchlorate); or a mixture of the 100% water or the solution and a from 1% to 5% organic solvent (e.g., acetonitrile).
In the case where a mobile phase containing water as its main component is used with a reversed-phase stationary phase to which an alkyl group having from 8 to 18 carbons is bonded, that is widely used as a stationary phase for a reversed-phase liquid chromatography, there are known those facts that time of retention of sample is not stable and the retention time decreases as the time in which the mobile phase is flowed increases, that is, that reproducibility of the retention time is low. In particular, in the case where the flow of the mobile phase is resumed after it is temporarily stopped, it is known that the retention time significantly decreases (see FIGS. 2, 3, and 5 shown in Patent Document 1 (i.e., Japanese Laid-Open Patent Document No. 2002-14086)). Since, in the chromatography, the compound separated is identified based on the retention time, the mobile phase cannot be used with the stationary phase if the reproducibility of retention time is low.
Conventionally, it has been speculated that the reason why the reproducibility of retention time is low is that when the mobile phase containing water as its main component is flowed through the stationary phase, the carbon chains gradually collapse due to their hydrophobicity and so-called “slipping” occurs, that is, the interaction between the stationary phase and the solute decreases. In order to solve the problem that the retention time decreases as the time increases, there are some cases where a column packing material having a stationary phase including a polar group, a column packing material having a stationary phase whose hydophobicity is lowered by decreasing the density of alkyl group, or a column packing material having a stationary phase that does not physically collapse, such as trimethylsilyl group, is used. The packing material having the stationary phase including the polar group may be a packing material including, as a stationary phase, a compound having a carbon chain and a high-polar group, such as amide group or carbamate group, bonded to the carbon chain, the stationary phase being bonded to a carrier (silica, polymer, etc.); or a packing material including stearyl group (its carbon number is 18) that is widely used as a hydrocarbon stationary phase, the stearyl group being bonded to a silica carrier, the residual silanol groups present on the silica carrier being decreased using a polar group as an endcapping.
In the above-described background, the Inventor had elucidated that, contrary to a conclusion expected from the above-explained slipping, the water-100 mobile phase can be used with a stationary phase including an alkyl group having a long carbon chain (whose carbon number is, e.g., 30) (see Patent Document 2 (i.e., Japanese Laid-Open Patent Document No. 2000-193648 or its corresponding U.S. Pat. No. 6,241,891)).
In addition, the Inventor had proved that, in the case where the pore diameter of a packing material carrying a stationary phase is large, or temperature is kept low, a mobile phase containing water as its main component can be used with either a stationary phase including an alkyl group having a long carbon chain or a stationary phase including an alkyl group having a short carbon chain whose carbon number is not greater than 22 (see the above-indicated Patent Document 1).
Thus, in the reversed-phase liquid chromatography in which the mobile phase containing water as its component is used, it is needed to use a column that is packed with a specific stationary phase usable with the mobile phase, or keep temperature low. However, since keeping the temperature low needs a cooling device, an additional expense is needed. In addition, the cost of each measurement is increased. Meanwhile, in the case where the temperature is not kept low, it is needed to use a column that is packed with a specific stationary phase, and accordingly a common reversed-phase column that is widely used in a reversed-phase liquid chromatography, such as a column packed with octadecyl group (whose carbon number is 18) that is most widely used, cannot be used because the common column has a high hydrophobicity.